209 MEAN SERU,A-IIAMUNGGLOBULINS (mg/dl) IN 19 PATIENTS WITH RHEUMATOID ARTHRITIS BEFORE AND AFTER LEVAMISOLE TREATMENT
stimulation of skin delayed-hypersensitivity tests was associated with depression of serum-immunoglobulins. This observation is consistent with the findings of Huskisson et al.3 A late de-
in antibody titres was observed by Renoux et al. in elderly levamisole-treated patients immunised with influenza crease
vaccine. Levamisole should therefore be considered rather as an immunomodulating agent than as a general immunostimulant. Yaron et al. gave an indication that levamisole can have an anti-inflammatory action independent of its effect on the immune system. We are tempted to suggest that further comparisons between the clinical effects of levamisole and immunoreactivity and intensity of inflammatory process in R.A. would make it possible to select patients who could benefit from this drug. H. SZPILMAN Institute of
Rheumatology,
Spartańska 1, 02-637
Warsaw, Poland
THE HEPARIN-THROMBIN CLOTTING-TIME
SIR,-The heparin-thrombin clotting-time (H.T.C.T.) is referred to in your exemplary editorial’ about antithrombin and the diagnosis of prethrombotic states. In case there’could be any confusion, I write to emphasise that this H.T.c.T. test is not influenced by antithrombin levels. Since we use the patient’s plasma as substrate, theoretically such an influence is possible; however, we accept an H.T.c.T. result only if a dilutethrombin clotting-time is strictly normal. You refer to this test as possibly useful in distinguishing between a diagnosis of acute myocardial infarction and "nonspecific chest pain". We continue to find the H.T.c.T. valuable in this situation, but the most striking abnormality in true infarction may not develop for two to four days. (The original studies were "between the first and the seventh day".) I think the H.T.c.T. may have greater value in detecting a prethrombotic state; the test has twice shown significantly shorter clotting-times in groups of patients studied long after various kinds of thrombosis who presumably are at greater risk than normal of further trouble_23 You do not mention that we think this test measures platelet factor 4 released from platelets and so reflects the degree of platelet activation. Portsmouth and South East Hampshire District Pathology Service, St.
Mary’s Hospital,
J. R. O’BRIEN
Portmouth PO3 6AG
S. LUFT D. GLIŃSKA-URBAN W. FISCHER M. PLACHECKA
LEVAMISOLE AS ADJUNCT TO DAPSONE IN LEPROSY
ALPHA-FETOPROTEIN ASSAY IN ALL AMNIOCENTESIS SAMPLES
SIR,-We envy Broch and Scrimgeour’s false-positive rate zero,8 and suspect we understand the reason. They regard an amniotic-fluid sample containing fetal blood as unusable, while our results9 simply reflect a total experience without prior sample selection. We do not feel that a sample contamiof
nated with fetal blood should be discarded since some 47% of fluids derived from pregnancies where the fetus had anencephaly contained fetal hoemoglobin.10 Since fetal blood seems to be present so often in these particular cases, repeat amniocenteses may be of no avail. We have lately been able to modify our assay, which is now more sensitive. Over 800 samples have been re-assayed, including the 15 (0-4) reported as "definite" false positives out of the 3536 cases studied.2 With the modified assay, only 5 of these now remain with A.F.P. values >3 S.D. above the mean. This suggests a very similar false-positive rate z 1%) to that reported in the European Medical Research Council report." Since we have not re-assayed all 3536 cases, our exact rate cannot now be stated, but it is clearly in the range where routine A.F.P. assays of all amniotic fluids would be justified. Even by declaring samples with fetal haemoglobin unusable, we predict that a zero false-positive rate will not be maintained. We offer ultrasound and amniographic studies as additional approaches when faced with borderline elevations of A.F.P., and do not rely solely on the A.F.P. value. Eunice
Kennedy
Shriver
Center,
altham, and Massachusetts General Hospital,
W
Boston, Massachusetts, U S A
6 Renoux, G .
AUBREY MILUNSKY MARGARET E. KIMBALL
Renoux, M., Morand, P., Dartigues, P. Rev. med. Tours, 1973,
7, 797. Yaron, I., Herzberg, M Lancer, Feb. 14, 1976, p. 369 H., Scrimgeour, J. B Lancet, 1976, i, 1404. 9 Milunsky, A . Alpert, E. ibid. 1976, i, 1015. 10 Milunsky, A.. Alpert, E Obstet Gynec. in the press . 11 Lindsten, J, Zetterstrom, R., Ferguson-Smith, M. in Prenatal Diagnosis of Genetic Disorders of the Fœtus. I N.S.E R.M., Paris, 1976. 7 Yaron, M. Brock, D J
8
SIR,-Levamisole has a stimulatory effect on some aspects of the cellular immunity mechanism of the host. The mechanism of action is unclear. It has been suggested that levamisole has a immunostimulatory effect in leprosy patients with depressed cell-mediated immunity.56 We have tried to find out if levamisole would affect the course of the disease in patients with lepromatous and dimorphic leprosy treated with dapsone. Patients with nodular lepromatous or nodular dimorphic leprosy took part in the study. These patients were receivingdapsone, by mouth or by injection. Patients were evaluated monthly for clinical lesions and reactions. Clinical lesions were graded zero or 1-4, 4 being a nodular lesion and zero being a totally flat lesion (macule). The same observer graded these lesions every time but the treatment was unknown to the observer. Smears were read and graded by the same laboratory personnel who did not know the patient’s name or treatment. Sixteen patients were treated with dapsone 25mg by mouth every day and four received 450mg acedapsone intramuscularly once every two months. These patients were advised to take an additional pill, which was either levamisole (150 mg) or placebo, one pill every two weeks. At the end of six months of therapy, 12 patients finished the trial from the 20 who had started. All 12 had been clinical grade 4at the start. The 6 who had been on levamisole in addition to their dapsone (or acedapsone) had lesions of 1+ or zero at the end of the trial. Of the 6 patients taking placebo 4 ended see with
as
-3 and 2
as
0. This is what
one
would expect
to
dapsone alone.
1 Lancet, 1976, i. 1333 2. O’Brien, J. R, Etherington, M. D., Jamieson, S, Klaber, M. R . Lincoln, S. V Thromb Diath. Hœmorrh 1974, 31, 279 3 O’Brien, J R., Etherington, M. D., Jamieson, S., Lawford, P., Lincoln, S V. Alkjaersig, N 4 Symoens, J. in Proceedings of the 2nd International Conference on Modulation of Host Resistance in the Prevention or Treatment of Induced Neoplasias Bethesda, 1975edited by M A Chirigos U S Government Printing Office in the press 5 Cardama, J E., Gatti, J. C., Balina, I. H . Cabrera, H N, Fliess, L L Int. J Leprosy, 1973, 41, 567. 6 Saint-Andre, P., Louvet, M. Med Arm. 1976, 4, 223.
J ibid 1975, 34, 483.
210 The prevalence of reactions before and after six months on levamisole or placebo was studied in 6 patients. All 6 had started out as 4+ but the 3 who were on placebo continued to have reactions (3+ or 4+) some necessitating the use of corticosteroids. The 3 levamisole-treated patients showed a gradual decrease of intensity of reactions to zero or +. When levamisole was withdrawn at the end of six months, one patient’s reactions increased again. The bacteriological index did not reflect any significant change between the placebo and the levamisole.
Leprology
of
of varicella in leukaemic children, live varicella vaccine would be a potent measure for prevention.
nature
Details of these children, their antibody responses and their before vaccination may be obtained from H.K.
Department of Pædiatrics Mie University School of Medicine, Tsu-City, Mie, Japan
Program,
Institute of Dermatology, Santa Domingo, Dominica
DENISE MARTINEZ
Department of Dermatology, Mount Sinai, Medical Center, Miami Beach, Florida, U.S A.
NARDO ZAIAS
immune
status
AKIHIKO HATTORI TOSHIAKI IHARA TOSHIAKI IWASA HITOSHI KAMIYA MINORU SAKURAI TADASU IZAWA
Research Institute for Microbial Diseases, Osaka University,
Suita, Osaka
MICHIAKI TAKAHASHI
CAN DOGS TRANSMIT HUMAN WARTS? USE OF LIVE VARICELLA VACCINE IN CHILDREN WITH ACUTE LEUKÆMIA OR OTHER
MALIGNANCIES
SiR,—Varicella infection is often severe or even fatal in children with leuk3emia or other malignancies. In a nationwide survey of leukaemic children in Japan we found that symptoms were severe in 56 out of 106 varicella-infected leukaemic children; 32 died. The risk of varicella has been increasing with the longer survival-times of these children and with the more intensive chemotherapy these children are given. Live varicella vaccine has proved safe and effective for children with chronic diseases such as nephrosis who had been receiving steroid therapy.’ We have tried this vaccine in children with acute leukxmia or other malignancies. Varicella vaccine (Oka strain passaged in human embryonic lung cells 11 times, guineapig embryo cells 12 times, and WI-38 cells 2-5 times) was given subcutaneously to 11 susceptible leukaemic children in remission. In the immunological check-up before vaccination, most of them showed positive reactions in the skin test with dinitrochlorobenzene, phytohaemagglutinin, purified protein derivative, and mumps and measles antigen. Leukopenia (three cases) and decreased IgG levels (two cases) were observed in some children. Anti-leukaemic medication was suspended from 1 week before vaccination to 1 week after vaccination. The only clinical reaction was a rash appearing 3 weeks after vaccination in two children, the rash disappeared within 3 days. Serological responses by complement fixing (C.F.) and neutralising (N.T.) tests were detected in all the vaccinated children at 4 weeks, and N.T. antibody was still detected 18 months after vaccination in the two cases tested. Two of the vaccinees were exposed to natural varicella in their classrooms 5 months and 2 months after vaccination, and one vaccinated child was exposed to varicella at home 18 months after vaccination. But they were free from any varicella symptoms. Five children with neuroblastoma or retinoblastoma were also vaccinated. Anti-cancer medication was suspended as for the leukaemic children. No clinical reactions were observed after vaccination and N.T. antibody response was observed in all of them. Follow-up of the vaccinated children is under way. Brunell et al.2reported that varicella could be prevented with zoster immune globulin when it was given within 72 h after exposure. In our experience, exposure is not noticed in most children until typical varicella has developed. Considering the increasing risk of exposure and the potentially fatal
SIR,-Antibodies against wart virus were studied, using the immunodiffusion method,’ in cattle, dog, and horse sera. Unexpectedly, antibodies were found in 23% of the dog sera. The frequency of antibodies in dogs was similar to that in children of the same age (table). Antibody titres were higher in sera from children over five years old. The highest titre found in dog sera was 16 and in the sera of children aged 1-12 years it was 32. 6 out of 70 cattle sera gave a faint precipitation line against human papilloma virus. The antibody titre in these 6 HUMAN WART-VIRUS ANTIBODY PREVALENCE IN CHILDREN AND DOGS
only 1. None of the 40 horse sera studied containe human wart-virus antibodies. In a retrospective investigation of the history of the dog and their owners’ families 2 out of 7 dogs with antibodies wer found to have had typical papillomas, oral in 1 and on the ski of its legs in the other; a third dog had a mammary tumour but it was not a typical papilloma histologically. The other dogs had no history of tumours. 1 of these 4 dogs without de tectable papillomas used to lick the neck of his owner. few months later the owner got typical warts in the exact plac which the dog licked. Upon examination of the dog one yea after the owner developed warts, no papillomas could be foun in the dog’s mouth. These findings reveal that human papilloma virus, or closely related virus, can infect dogs and, sometimes, catt also. The virus extracted from dog papillomas is antigenical completely divergent from human wart virus.2 It would be teresting to know, therefore, whether the human wart vir can also induce papillomas in dogs or whether it is a passeng virus in dogs with subclinical or undetectable symptoms. The species specificity for the papovaviruses may not be clear cut as has been thought. Dogs-and, perhaps, catt also-must be considered as potential transmitters of warts
sera was
,
rnptn
Laboratory of Viral Immunopathology, Department of Virology, University of Helsinki, Helsinki 29, SF - 00290 Finland
1 2.
Takahashi, M., Otsuka, T., Okuno, Y., Asano, Y., Yazaki, T., Isomura, S., Lancet, 1974, ii, 1288. Brunell, P. A., Ross, A., Miller, L. H., Kuo, B. New Engl J. Med. 1969, 280, 1191.
3. Gershon, A. A ,
Steinberg, S., Brunell,
P. A ibid.
1974, 290, 243
SEPPO PYRHÖ
Pyrhönen, S., Penttinen, K. Lancet, 1972, ii, 1330. 2. Le Bouvier, G. L., Sussman, M., Crawford, L. V J. gen. Microbiol 19 1.
45, 497.
stimulation of skin delayed-hypersensitivity tests was associated with depression of serum-immunoglobulins. This observation is consistent with the findings of Huskisson et al.3 A late de-
in antibody titres was observed by Renoux et al. in elderly levamisole-treated patients immunised with influenza crease
vaccine. Levamisole should therefore be considered rather as an immunomodulating agent than as a general immunostimulant. Yaron et al. gave an indication that levamisole can have an anti-inflammatory action independent of its effect on the immune system. We are tempted to suggest that further comparisons between the clinical effects of levamisole and immunoreactivity and intensity of inflammatory process in R.A. would make it possible to select patients who could benefit from this drug. H. SZPILMAN Institute of
Rheumatology,
Spartańska 1, 02-637
Warsaw, Poland
THE HEPARIN-THROMBIN CLOTTING-TIME
SIR,-The heparin-thrombin clotting-time (H.T.C.T.) is referred to in your exemplary editorial’ about antithrombin and the diagnosis of prethrombotic states. In case there’could be any confusion, I write to emphasise that this H.T.c.T. test is not influenced by antithrombin levels. Since we use the patient’s plasma as substrate, theoretically such an influence is possible; however, we accept an H.T.c.T. result only if a dilutethrombin clotting-time is strictly normal. You refer to this test as possibly useful in distinguishing between a diagnosis of acute myocardial infarction and "nonspecific chest pain". We continue to find the H.T.c.T. valuable in this situation, but the most striking abnormality in true infarction may not develop for two to four days. (The original studies were "between the first and the seventh day".) I think the H.T.c.T. may have greater value in detecting a prethrombotic state; the test has twice shown significantly shorter clotting-times in groups of patients studied long after various kinds of thrombosis who presumably are at greater risk than normal of further trouble_23 You do not mention that we think this test measures platelet factor 4 released from platelets and so reflects the degree of platelet activation. Portsmouth and South East Hampshire District Pathology Service, St.
Mary’s Hospital,
J. R. O’BRIEN
Portmouth PO3 6AG
S. LUFT D. GLIŃSKA-URBAN W. FISCHER M. PLACHECKA
LEVAMISOLE AS ADJUNCT TO DAPSONE IN LEPROSY
ALPHA-FETOPROTEIN ASSAY IN ALL AMNIOCENTESIS SAMPLES
SIR,-We envy Broch and Scrimgeour’s false-positive rate zero,8 and suspect we understand the reason. They regard an amniotic-fluid sample containing fetal blood as unusable, while our results9 simply reflect a total experience without prior sample selection. We do not feel that a sample contamiof
nated with fetal blood should be discarded since some 47% of fluids derived from pregnancies where the fetus had anencephaly contained fetal hoemoglobin.10 Since fetal blood seems to be present so often in these particular cases, repeat amniocenteses may be of no avail. We have lately been able to modify our assay, which is now more sensitive. Over 800 samples have been re-assayed, including the 15 (0-4) reported as "definite" false positives out of the 3536 cases studied.2 With the modified assay, only 5 of these now remain with A.F.P. values >3 S.D. above the mean. This suggests a very similar false-positive rate z 1%) to that reported in the European Medical Research Council report." Since we have not re-assayed all 3536 cases, our exact rate cannot now be stated, but it is clearly in the range where routine A.F.P. assays of all amniotic fluids would be justified. Even by declaring samples with fetal haemoglobin unusable, we predict that a zero false-positive rate will not be maintained. We offer ultrasound and amniographic studies as additional approaches when faced with borderline elevations of A.F.P., and do not rely solely on the A.F.P. value. Eunice
Kennedy
Shriver
Center,
altham, and Massachusetts General Hospital,
W
Boston, Massachusetts, U S A
6 Renoux, G .
AUBREY MILUNSKY MARGARET E. KIMBALL
Renoux, M., Morand, P., Dartigues, P. Rev. med. Tours, 1973,
7, 797. Yaron, I., Herzberg, M Lancer, Feb. 14, 1976, p. 369 H., Scrimgeour, J. B Lancet, 1976, i, 1404. 9 Milunsky, A . Alpert, E. ibid. 1976, i, 1015. 10 Milunsky, A.. Alpert, E Obstet Gynec. in the press . 11 Lindsten, J, Zetterstrom, R., Ferguson-Smith, M. in Prenatal Diagnosis of Genetic Disorders of the Fœtus. I N.S.E R.M., Paris, 1976. 7 Yaron, M. Brock, D J
8
SIR,-Levamisole has a stimulatory effect on some aspects of the cellular immunity mechanism of the host. The mechanism of action is unclear. It has been suggested that levamisole has a immunostimulatory effect in leprosy patients with depressed cell-mediated immunity.56 We have tried to find out if levamisole would affect the course of the disease in patients with lepromatous and dimorphic leprosy treated with dapsone. Patients with nodular lepromatous or nodular dimorphic leprosy took part in the study. These patients were receivingdapsone, by mouth or by injection. Patients were evaluated monthly for clinical lesions and reactions. Clinical lesions were graded zero or 1-4, 4 being a nodular lesion and zero being a totally flat lesion (macule). The same observer graded these lesions every time but the treatment was unknown to the observer. Smears were read and graded by the same laboratory personnel who did not know the patient’s name or treatment. Sixteen patients were treated with dapsone 25mg by mouth every day and four received 450mg acedapsone intramuscularly once every two months. These patients were advised to take an additional pill, which was either levamisole (150 mg) or placebo, one pill every two weeks. At the end of six months of therapy, 12 patients finished the trial from the 20 who had started. All 12 had been clinical grade 4at the start. The 6 who had been on levamisole in addition to their dapsone (or acedapsone) had lesions of 1+ or zero at the end of the trial. Of the 6 patients taking placebo 4 ended see with
as
-3 and 2
as
0. This is what
one
would expect
to
dapsone alone.
1 Lancet, 1976, i. 1333 2. O’Brien, J. R, Etherington, M. D., Jamieson, S, Klaber, M. R . Lincoln, S. V Thromb Diath. Hœmorrh 1974, 31, 279 3 O’Brien, J R., Etherington, M. D., Jamieson, S., Lawford, P., Lincoln, S V. Alkjaersig, N 4 Symoens, J. in Proceedings of the 2nd International Conference on Modulation of Host Resistance in the Prevention or Treatment of Induced Neoplasias Bethesda, 1975edited by M A Chirigos U S Government Printing Office in the press 5 Cardama, J E., Gatti, J. C., Balina, I. H . Cabrera, H N, Fliess, L L Int. J Leprosy, 1973, 41, 567. 6 Saint-Andre, P., Louvet, M. Med Arm. 1976, 4, 223.
J ibid 1975, 34, 483.
210 The prevalence of reactions before and after six months on levamisole or placebo was studied in 6 patients. All 6 had started out as 4+ but the 3 who were on placebo continued to have reactions (3+ or 4+) some necessitating the use of corticosteroids. The 3 levamisole-treated patients showed a gradual decrease of intensity of reactions to zero or +. When levamisole was withdrawn at the end of six months, one patient’s reactions increased again. The bacteriological index did not reflect any significant change between the placebo and the levamisole.
Leprology
of
of varicella in leukaemic children, live varicella vaccine would be a potent measure for prevention.
nature
Details of these children, their antibody responses and their before vaccination may be obtained from H.K.
Department of Pædiatrics Mie University School of Medicine, Tsu-City, Mie, Japan
Program,
Institute of Dermatology, Santa Domingo, Dominica
DENISE MARTINEZ
Department of Dermatology, Mount Sinai, Medical Center, Miami Beach, Florida, U.S A.
NARDO ZAIAS
immune
status
AKIHIKO HATTORI TOSHIAKI IHARA TOSHIAKI IWASA HITOSHI KAMIYA MINORU SAKURAI TADASU IZAWA
Research Institute for Microbial Diseases, Osaka University,
Suita, Osaka
MICHIAKI TAKAHASHI
CAN DOGS TRANSMIT HUMAN WARTS? USE OF LIVE VARICELLA VACCINE IN CHILDREN WITH ACUTE LEUKÆMIA OR OTHER
MALIGNANCIES
SiR,—Varicella infection is often severe or even fatal in children with leuk3emia or other malignancies. In a nationwide survey of leukaemic children in Japan we found that symptoms were severe in 56 out of 106 varicella-infected leukaemic children; 32 died. The risk of varicella has been increasing with the longer survival-times of these children and with the more intensive chemotherapy these children are given. Live varicella vaccine has proved safe and effective for children with chronic diseases such as nephrosis who had been receiving steroid therapy.’ We have tried this vaccine in children with acute leukxmia or other malignancies. Varicella vaccine (Oka strain passaged in human embryonic lung cells 11 times, guineapig embryo cells 12 times, and WI-38 cells 2-5 times) was given subcutaneously to 11 susceptible leukaemic children in remission. In the immunological check-up before vaccination, most of them showed positive reactions in the skin test with dinitrochlorobenzene, phytohaemagglutinin, purified protein derivative, and mumps and measles antigen. Leukopenia (three cases) and decreased IgG levels (two cases) were observed in some children. Anti-leukaemic medication was suspended from 1 week before vaccination to 1 week after vaccination. The only clinical reaction was a rash appearing 3 weeks after vaccination in two children, the rash disappeared within 3 days. Serological responses by complement fixing (C.F.) and neutralising (N.T.) tests were detected in all the vaccinated children at 4 weeks, and N.T. antibody was still detected 18 months after vaccination in the two cases tested. Two of the vaccinees were exposed to natural varicella in their classrooms 5 months and 2 months after vaccination, and one vaccinated child was exposed to varicella at home 18 months after vaccination. But they were free from any varicella symptoms. Five children with neuroblastoma or retinoblastoma were also vaccinated. Anti-cancer medication was suspended as for the leukaemic children. No clinical reactions were observed after vaccination and N.T. antibody response was observed in all of them. Follow-up of the vaccinated children is under way. Brunell et al.2reported that varicella could be prevented with zoster immune globulin when it was given within 72 h after exposure. In our experience, exposure is not noticed in most children until typical varicella has developed. Considering the increasing risk of exposure and the potentially fatal
SIR,-Antibodies against wart virus were studied, using the immunodiffusion method,’ in cattle, dog, and horse sera. Unexpectedly, antibodies were found in 23% of the dog sera. The frequency of antibodies in dogs was similar to that in children of the same age (table). Antibody titres were higher in sera from children over five years old. The highest titre found in dog sera was 16 and in the sera of children aged 1-12 years it was 32. 6 out of 70 cattle sera gave a faint precipitation line against human papilloma virus. The antibody titre in these 6 HUMAN WART-VIRUS ANTIBODY PREVALENCE IN CHILDREN AND DOGS
only 1. None of the 40 horse sera studied containe human wart-virus antibodies. In a retrospective investigation of the history of the dog and their owners’ families 2 out of 7 dogs with antibodies wer found to have had typical papillomas, oral in 1 and on the ski of its legs in the other; a third dog had a mammary tumour but it was not a typical papilloma histologically. The other dogs had no history of tumours. 1 of these 4 dogs without de tectable papillomas used to lick the neck of his owner. few months later the owner got typical warts in the exact plac which the dog licked. Upon examination of the dog one yea after the owner developed warts, no papillomas could be foun in the dog’s mouth. These findings reveal that human papilloma virus, or closely related virus, can infect dogs and, sometimes, catt also. The virus extracted from dog papillomas is antigenical completely divergent from human wart virus.2 It would be teresting to know, therefore, whether the human wart vir can also induce papillomas in dogs or whether it is a passeng virus in dogs with subclinical or undetectable symptoms. The species specificity for the papovaviruses may not be clear cut as has been thought. Dogs-and, perhaps, catt also-must be considered as potential transmitters of warts
sera was
,
rnptn
Laboratory of Viral Immunopathology, Department of Virology, University of Helsinki, Helsinki 29, SF - 00290 Finland
1 2.
Takahashi, M., Otsuka, T., Okuno, Y., Asano, Y., Yazaki, T., Isomura, S., Lancet, 1974, ii, 1288. Brunell, P. A., Ross, A., Miller, L. H., Kuo, B. New Engl J. Med. 1969, 280, 1191.
3. Gershon, A. A ,
Steinberg, S., Brunell,
P. A ibid.
1974, 290, 243
SEPPO PYRHÖ
Pyrhönen, S., Penttinen, K. Lancet, 1972, ii, 1330. 2. Le Bouvier, G. L., Sussman, M., Crawford, L. V J. gen. Microbiol 19 1.
45, 497.
